As known in the art, various techniques have been used to determine whether a chemical interaction has occurred between two materials, such as between a probe carrying a binding partner and a sample. In the specific example of determining whether DNA hybridization has occurred, various techniques have been used to extract information from a sample. For example, detection schemes have been used that are responsive to fluorescence in order to reveal specific interactions or hybridizations. U.S. Pat. No. 5,578,832, "Method and Apparatus for Imaging a Sample on a Device," issued to Trulson et al. ("the '832 patent") and U.S. Pat. No. 5,631,734, "Method and Apparatus for Detection of Fluorescently Labeled Materials," issued to Stern et al. ("the '734 patent") provide methods and systems for detecting a labeled marker on a sample located on a support through the use of an excitation radiation source and radiation optics. The '832 patent and the '734 patent are hereby incorporated by reference for all they disclose and for all purposes. As described in the '832 and '734 patents, these techniques employ the use of a label, for example, the DNA probe is labeled with a fluorescent molecule, such as fluorophore or biotin. Once the DNA probe is labeled according to prior methods, an optical system can be used to determine whether hybridization has occurred by measuring fluorescence activated between the labeled sample and the probe material.
The present invention provides a method for determining whether a chemical interaction has occurred between naturally occurring bio-polymers which are non-identical binding partners through detecting a physical or chemical change on a micromechanical device called a cantilever. A cantilever, by way of analogy, can be thought of as a diving board which has been reduced to a very small size. More specifically, a cantilever is a physical device that is attached to another object at one end and remains free to move on the other end. Deflection or up and down movement of the free end of the cantilever can then be detected. The method of the present invention can be used with any chemical analyte to generate a physical or chemical change, whether through affinity binding, which may include hydrogen bonding, electrostatic attractions, hydrophobic effects, dipole interactions, or through other forces.
The use of micromechanical sensors is advantageous in the method of the present invention for several reasons. Various signals such as force, heat, stress, magnetism, charge, radiation and chemical reactions can be readily transduced into a micromechanical deflection by an appropriately coated structure, such as a cantilever. In addition, silicon-based micromechanical devices can easily be integrated into microelectronic processing systems such as CMOS (Complementary Metal-Oxide-Semiconductor), as known to one of skill in the art. As a result, it is possible to produce seamless sensors as low cost and to integrate them directly into computers. Moreover, micromechanical sensors are very small, typically approximately 400 .mu.m in length, approximately 40 .mu.m wide and approximately 1 .mu.m thick. As a result, it is possible to obtain a short response time, generally measured in microseconds, as well as sensitivity superior to standard techniques. Finally, it is possible to construct arrays of micromechanical devices, thereby permitting complex analysis of a variety of signals as well as the use of a variety of sensing materials.
By way of background, it is known that stress induced by self-assembled monolayers can be detected by observing the deflection of a micromachined cantilever similar to those used in the commercial Atomic Force Microscope ("AFM"), as described by Berger et al., in "Surface Stress in the Self-Assembly of Alkanethiols on Gold," Science, Jun. 27, 1997, Vol. 176, p. 2021 ("Berger I"), which is hereby incorporated by reference for all it teaches. The Berger et al. paper studied the surface stress changes during self-assembly of selected molecules, including alkanethiol molecules self-assembled on gold. The researchers found that the stress increases linearly with the length of the alkyl chain of the molecule. In addition, the researchers detected a change in the state of stress with the formation of salt bridges formed when mercaptohexadecanoic acid was deposited on a functionalized surface coated with the self-assembled thiols. This change in cantilever stress was used to detect the formation of the salt bridges when the analyte molecules were introduced.
Other pertinent work involving micromechanical sensors is reflected in a paper by Berger et al. entitled "Nanometers, Picowatts, Femtojoules: Thermal Analysis and Optical Spectroscopy Using Micromechanics," Analytical Methods & Instrumentation, Special Issue, .mu.TAS '96 ("Berger II"), also incorporated by reference for all it discloses and for all purposes. In Berger II, examples of low-cost, disposable micromechanical devices are described which perform optical absorption spectra, calorimetric and thermal analysis, electrochemical stressograms, gas phase adsorption and surface reaction monitors.
Other work in the area of micromechanical sensors is reported by Gimzewski et al. in "Observation of a chemical reaction using a micromechanical sensor," Chemical Physics Letters, Vol. 217, No. 5,6, Jan. 28, 1994, ("Gimzewski") which is hereby incorporated by reference for all it discloses and for all purposes. Gimzewski discloses a calorimeter for sensing chemical reactions. The device is based on a micromechanical silicon lever coated with a layer of aluminum. A sample is deposited on the lever in a thin layer. Heat fluxes are detected by measuring the deflection of the cantilever induced by the differential thermal expansion of the lever. Specifically, Gimzewski discloses using this technique to review the catalytic conversion of H.sub.2 +O.sub.2 to obtain H.sub.2 O.
It is further known to operate multiple probes for the atomic force microscope. As described by Minne et al., "Automated parallel high-speed atomic force microscopy," Applied Physics Letters, Volume 78, No. 18, May 4, 1998 ("Minne"), which is herein incorporated by reference for all it discloses and for all purposes, an expandable system is provided to operate multiple probes for the atomic force microscope in parallel at high speeds. The cantilever footprint is only 200 .mu.m wide which allows the devices to be placed in a one-dimensional expandable parallel array.
Yet another contribution to the art of micromechanical sensors is described by Manalis et al., "Interdigital cantilevers for atomic force microscopy," Applied Physics Letters, Vol. 69, No. 25, Dec. 16, 1996 ("Manalis I"), which is hereby incorporated by reference for all it discloses and for all purposes. In Manalis I, an AFM sensor is described in which a silicon cantilever is micromachined into the shape of interdigitated fingers that form a diffraction grating. When detecting a force, alternating fingers are displaced while remaining fingers are held fixed. As a result, a phase sensitive diffraction grating is created which allows the cantilever displacement to be determined by measuring the intensity of diffracted modes.
Another paper by Lang et al., "Sequential position readout from arrays of micromechanical cantilever sensors," Applied Physics Letters, Vol. 73, p. 383, 1998 ("Lang") describes using a reference cantilever for canceling environmental noise. Lang is hereby incorporated by reference for all it discloses and for all purposes. In Lang, chemically specific responses are extracted in a noisy environment using a sensor to detect specific chemical interactions and an uncoated cantilever as a reference.
Finally, another paper by Manalis et al., "Two dimensional micromechanical bimorph arrays for detection of thermal radiation," Applied Physics Letters, Jun. 16, 1997, (Manalis II) hereby incorporated by reference for all it discloses and for all purposes, describes fabricating arrays of cantilevers and using them as sensitive detectors of head induced stress. Specifically, the cantilevers described by Manalis II were placed on a grid with 50 microns on centers. The present inventors have determined that this type of array is a suitable substrate for determining, for example, hybridization.